Kaposi's sarcoma (KS) remains as an important cancer in AIDS patients despite anti-retroviral therapy. It is the most common cancer in both HIV-positive and -negative populations in many African countries. Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication is essential for KS development, and is an effective prognostic marker for KS. In the current funding period, we have identified ERK, p38 and JNK mitogen- activated protein kinase (MAPK) pathways as the essential mediators of KSHV lytic replication. Furthermore, we have shown that reactive oxygen species (ROS) hydrogen peroxide is essential and sufficient for activating KSHV lytic replication. Significantly, ROS scavengers such as antioxidant N-acetyl-L-cysteine (NAC) inhibit KSHV replication in vitro and in a primary effusion lymphoma (PEL) mouse model, and slows the progression of lymphoma. The objective of this application is to further dissect the molecular mechanism by which MAPK pathways and ROS mediate KSHV lytic replication induced by physiological triggers including inflammatory cytokines and hypoxia, and examine the preventive and therapeutic effect of targeting ROS for inhibiting KSHV replication and KS development. The central hypothesis is that inflammatory cytokines and hypoxia activate MAPK pathways and induce ROS, resulting in the activation of KSHV lytic replication, and as a result, ROS scavengers can effectively inhibit KSHV lytic replication and KS development. We have recently developed a novel KSHV-induced KS model, which is particularly useful for preclinical testing of novel agents targeting KSHV lytic replication and KS development. We will test the hypothesis by performing the following four Specific Aims: 1) To examine the individual and combined effects of hypoxia and inflammatory cytokines on activating KSHV lytic replication, and the involvement of ROS and MAPK pathways; 2) To define the molecular interactions of ROS and MAPK pathways that mediate KSHV lytic replication induced by hypoxia and inflammation cytokines; 3) To define KSHV epigenetic signatures in MAPK pathways-mediated viral lytic replication induced by ROS, inflammation cytokines and hypoxia; and 4) To examine the preventive and therapeutic effect of ROS scavengers on KSHV lytic replication and KS development in a novel KSHV-induced KS model. The proposed project is highly significant because it will define the molecular basis underlying KSHV lytic replication induced by physiological triggers, and identify novel intervention targets for KSHV- induced malignancies. It will employ innovative contemporary technologies and a novel KSHV-induced KS model. We expect to illustrate an essential role of ROS and MAPK pathways in KSHV lytic replication, and demonstrate efficient inhibition of KSHV lytic replication and KS development by ROS scavenger NAC, which should establish the basis for future clinical trials for KSHV-induced malignancies in HIV patients. Because of their wide availability and affordability, NAC and other antioxidants are attractive agents for KSHV-associated malignancies, particularly in underserved populations and in Africa.